Collagen proteins in connective tissues constitute a three-dimensional network with crosslinks. The crosslinks are formed after deposition of the collagen molecule in the extracellular matrix. Different types of crosslinks between collagen molecules are known. As a result from enzymatic reactions, lysino-norleucine-type difunctional crosslinks, pyridinoline-type trifunctional crosslinks, and pyrrole-type crosslinks are formed. Aberrant degradation of collagen is an indication of disorders of connective tissues. Disorders of bone tissue comprise osteoporosis, Paget's disease, bone tumours, and drug-related bone loss. Disorders of cartilage tissue occur in arthritis (such as osteo-arthritis and rheumatoid arthritis), and disorders of the cardiovascular system are prominent in atherosclerosis, aneurisms, hypertension, myocardial infarction and hypertrophy. Similarly, disorders of the respiratory system and liver, such as fibrosis, of the teeth and periodontal tissues, of skin and wound healing, and in tumours involve changes in collagen turnover.
Known methods to determine turnover of connective tissues, in particular bone, are based on serum/plasma/blood or urinary levels of pyridinoline crosslinks (free or still containing residual amino acids), or telopeptide sequences corresponding to the crosslinking site. For example U.S. Pat. No. 5,140,103 discloses a method for measuring collagen degradation by quantitating the concentration of a C-terminal collagen type II or III telopeptide containing a pyridinoline-type crosslink by immunological, electrochemical or fluorometric assays. WO-92/21698 describes a method of determining bone resorption by means of antibodies to type I collagen telopeptides. WO-95/08115 discloses a method for assaying collagen fragments by means of antibodies against crosslinking sites of collagen. Similarly, WO-91/10141 describes the use of antibodies to detect collagen-derived crosslinks in biological fluids as a diagnostic method of bone diseases.
A disadvantage of these known methods for assaying collagen degradation is that the detection means (e.g. antibodies) recognise an epitope that includes the crosslink, or an epitope close to the crosslinking site. Thus, these methods depend on the nature and structure of the crosslink, or the amino acid sequence close to the crosslinking site. Collagen fragments containing other types of crosslinks are not necessarily detected and thus not quantitated, which leads to a less reliable and/or less sensitive result. More importantly, most of these methods require a relatively insensitive and laborious competition-type immunoassay that is mostly only applicable to urine, and not to serum/plasma/blood. Disadvantages of urinary assays are the need to correct for the urine volume based on creatinine levels, diurnal variation in urinary excretion rate and the need to collect urine samples.